RNA viruses are known to evolve rapidly in nature. Emergence of new virus strains with altered virulence and tissue and host specificity is an important consequence of virus evolution. RNA recombination, one of the critical forces in virus evolution results in genome rearrangements and formation of chimeric RNA genomes. Sindbis virus (SIN) is a mosquito transmitted human RNA virus. Many encephalitis viruses including Rubella virus are related to Sindbis virus. It was previously demonstrated that two SIN RNAs that were transfected into animal cells in culture can undergo recombination leading to production of live virus. In continuation of this study, the sequence and structural requirements of the participating templates will be explored using engineered RNA transcripts. This study specifically aims to answer the following questions: 1) What is the role of 3' and 5' non-viral sequences in recombination? 2) What are the sequences at the cross-over sites of recombinant RNAs. 3) Is there a sequence heterogeneity at the site of cross-over? To answer these questions, RNA templates with appropriate modifications will be synthesized from corresponding plasmids. These RNAs will be allowed to undergo recombination in vivo, and the progeny RNAs will be analyzed by electrophoresis, PCR mediated sequencing of cDNAs, and analysis of the resulting sequences. These studies are expected to aid in the development of genetically engineered RNA viral vaccines.